Crystal Structure of a MarR Family Polypeptide

ABSTRACT

The crystal structure of the product, crystals of the MarR protein, a regulator of multiple antibiotic resistance in  Escherichia coli , and methods of crystallization of the MarR protein are described.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/707,404, filed Feb. 16, 2007; which is a continuation applicationof U.S. application Ser. No. 10/196,655, filed Jul. 15, 2002, now U.S.Pat. No. 7,202,339, issued Apr. 10, 2007; which claims priority to U.S.Provisional Patent Application Ser. No. 60/388,622, filed on Jun. 13,2002; and U.S. Provisional Patent Application Ser. No. 60/305,404, filedon Jul. 13, 2001. The entire contents of all of the above applicationsand patents are hereby incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under GM51661 awarded byThe National Institutes of Health. The government may, therefore, havecertain rights in the invention.

BACKGROUND OF THE INVENTION

The Mar phenotype in E. coli is attributed largely to the action ofMarA, the expression of which is regulated by MarR (Alekshun, M. N.supra (1997)). MarA is a transcription factor that autoactivatesexpression of the marRAB operon and regulates the expression of a globalnetwork of more than 60 chromosomal genes (Martin, R. G. et al. J. Bact.178, 2216-2223 (1996); Barbosa, T. M. & Levy, S. B. J. Bact. 182,3467-3474 (2000)). Mar mutants in isolates of clinical origin have nowbeen identified (Maneewannakul, K. & Levy, S. B. Antimicrob. AgentsChemother. 40, 1695-1698 (1996); Oethinger, M. et al. Antimicrob. AgentsChemother. 42, 2089-2094 (1998); Linde, H. J. et al. Antimicrob. AgentsChemother. 44, 1865-1868 (2000); Ziha-Zarifi, I., et al. Antimicrob.Agents Chemother. 43, 287-291 (1999); Koutsolioutsou, A et al.Antimicrob. Agents Chemother. 45, 38-43 (2001)). Constitutiveoverexpression of MarA or a MarA homolog in many of these strains is akey contributor to the maintenance of the resistance phenotype,particularly with respect to the fluoroquinolones, and recent studieshave documented the selection of Mar mutants, bearing mutations in MarR,MexR, or other homologous loci, in E. coli, Pseudomonas aeruginosa, andother organisms during antimicrobial chemotherapy (Oethinger, M supra;Linde, H. J. et al.; supra; Ziha-Zarifi, I. et al. supra; Kern, W. V.,et al. Antimicrob. Agents Chemother. 44, 814-820 (2000)).

MarR is a regulator of multiple antibiotic resistance in Escherichiacoli. It is the prototypic member of a family of regulatory proteinsfound in the Bacteria and the Archaea that play important roles in thedevelopment of antibiotic resistance, a global health problem. In theabsence of an appropriate stimulus, MarR negatively regulates expressionof the marRAB operon (Cohen, S. P., et al. 1993. J. Bacteriol. 175:1484-1492; Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad. Sci.92: 5456-5460; Seoane, A. S. and Levy, S. B. 1995. J. Bacteriol. 177:3414-3419, 1995). DNA footprinting experiments suggest that MarRdimerizes at two locations, sites I and II, health problem. In theabsence of an appropriate stimulus, MarR negatively regulates expressionof the marRAB operon (Cohen, S. P., et al. 1993. J. Bacteriol. 175:1484-1492; Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad. Sci.92: 5456-5460; Seoane, A. S. and Levy, S. B. 1995. J. Bacteria 177:3414-3419, 1995). DNA footprinting experiments suggest that MarRdimerizes at two locations, sites I and II, within the mar operator(marO) (Martin and Rosner, 1995, supra). Site I is positioned among the−35 and −10 hexamers and site II spans the putative MarR ribosomebinding site (reviewed in Alekshun, M. N. and Levy, S. B. 1997.Antimicrob. Agents Chemother. 10: 2067-2075).

MarR is a member of a newly recognized family of regulatory proteins(Alekshun, M. N. and Levy, S. B. 1997. Antimicrob. Agents Chemother. 10:2067-2075. Sulavik, M. C., et al. 1995. Mol. Med. 1: 436-446) and manyfunctional homologues have been identified in a variety of importanthuman pathogens and have been found to regulate a variety of differentprocesses. For example, some MarR homologues have been found to controlexpression of multiple antibiotic resistance operons, some regulatetissue-specific adhesive properties, some control expression of acryptic hemolysin, some regulate protease production, and some regulatesporulation. Proteins of the MarR family control an assortment ofbiological functions including resistance to multiple antibiotics,organic solvents, household disinfectants, and oxidative stress agents,collectively termed the multiple antibiotic resistance (Mar) phenotype(Alekshun, M. N. & Levy, S. B. Trends Microbiol. 7, 410-413 (1999)).These proteins also regulate the synthesis of pathogenic factors inmicrobes that infect humans and plants (Miller, P. F. & Sulavik, M. C.Mol. Microbiol. 21, 441-448 (1996)). Insight into the three dimensionalstructure of MarR family proteins would be of great value in designingdrugs that interact with this family of proteins and modulate MarRfunction, for example, antibiotic resistance and virulence.

SUMMARY OF THE INVENTION

The instant invention advances the prior art by providing the crystalstructure of a MarR family polypeptide, MarR. The crystal structure wassolved for both the MarR polypeptide and the MarR polypeptide bound tosalicylate.

The invention pertains at least in part to a crystallized MarR familypolypeptide. The MarR family polypeptide is crystallized underappropriate conditions such that its three dimensional structure can bedetermined. In another embodiments, the crystallized MarR familypolypeptide is given by the atomic coordinates given in FIG. 7 or 8.

In another embodiment, the invention pertains, at least in part, to amethod for determining the three dimensional structure of MarR familypolypeptide. The method includes crystallizing the MarR familypolypeptide under appropriate conditions such that crystals are formed,and analyzing it, such that its three dimensional structure isdetermined.

In yet another embodiment, the invention pertains to a method of makinga crystal of a MarR family polypeptide. The method includes contactingMarR with a modulator to form a complex and allowing crystals of thecomplex to grow, e.g., by using hanging droplet vapor diffusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence alignment of MarR with representative membersof the MarR family (SEQ ID NOS 2-8, respectively, in order ofappearance).

FIG. 2 is a ribbon representation of the cocrystal structure of the MarRdimer with salicylate viewed with the subunit two-fold axis nearvertical.

FIG. 3 is an electrostatic surface representation of the MarR dimer.

FIG. 4 is a Cα trace of a MarR subunit in stereo representation.

FIG. 5 is a representation of the N-/C-terminal domain represented by asurface around the van der Waals radii of the side chain atoms only ofthe hydrophobic core residues. Helices leading to and from the domainare shown in ribbon representation.

FIG. 6 is a diagram which shows interactions between the DNA-bindingdomains of the dimer in the region of the Arg 73-Asp 67′ salt bridges.The stereo view is coincident with the 2-fold rotation axis of thedimer. Electron density shown is a 2F_(O)-F_(C) map contoured at 1σ.

FIG. 7 shows the atomic coordinates of the MarR-salicylate co-crystal(residues 7-144 of SEQ ID NO:2).

FIG. 8 shows the atomic coordinates of the MarR crystal withoutsalicylate (residues 12-144 and 9-144 of SEQ ID NO:2).

FIG. 9 is a ribbon representation of the MarR dimer with the two-foldaxis near vertical.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains, at least in part, to crystallized MarR familypolypeptides. The crystallized MarR family polypeptides are crystallizedunder appropriate conditions such that the three dimensional structurecan be determined using methods described herein and/or art recognizedtechniques.

The term “MarR family polypeptide” includes molecules related to MarR,e.g., having certain shared structural and functional features. MarRfamily polypeptides also include those which are structural homologs ofMarR. The structural homologs include those having a crystallized formwhich are structurally similar to that of crystallized MarR. MarR familymembers, in addition to having similarity to MarR, may bind to DNA andregulate transcription. While some MarR family members negativelycontrol transcription (e.g., MarR), others have positive/activatorfunctions (e.g., SlyA, BadR, NhhD, and MexR). MarR family polypeptidescomprise DNA and protein binding domains. In addition, MarR familypolypeptides can interact with a variety of structurally unrelatedcompounds that regulate their activity.

Exemplary MarR family members are taught in the art and can be found,e.g., in Sulavik et al. (1995. Molecular Medicine. 1:436), Miller andSulavik (1996. Molecular Microbiology. 21:441) in which alignments ofMarR and related proteins are shown, or through the use of BLASTsearches and other techniques known in the art. Exemplary MarR familypolypeptides are also illustrated in the following chart:

MarR Family Polypeptides Gram-negative Escherichia coli MarR SlyA EmrR(MprA) PapX PrsX HpcR Ec17 kD Salmonella typhimurium MarR SlyA EmrRPseudomonas aeruginosa MexR Erwinia chrysanthemi PecS Rhodopseudomonaspalustris BadR Burkholderia pseudomallei OrfE Gram-positive Bacillussubtilus YdcH YhbI YkmA YkoM Orf7 YfiV YetL YdgJ YwoH YwaE YwhA Hpr YybAYxaD YsmB YusO YpoP YkvE Bacillus firmus Orf7 Staphylococcus sciuriOrf145 Orf141 Butyrivibrio fibrisolvens CinR Sphingomonasaromaticivorans Orf158 Rhodococcus rhodochrous NhhD Streptomycespeucetius Orf1 Acid-fast Mycobacterium tuberculosis 14.7 kD Rv1404Rv0737 Rv0042c Yz08 (15.6 kD) Mycobacterium leprae Yz08 (15.6 kD)Archaea Methanobacterium thermoautotrophicum MTH313 Sulfolobussolfataricus Lrs14 Archaeoglobus fulgidus CinR Purple non-sulfurRhodobacter capsulatus PetP Sinorhizobium meliloti SlyA (E293909)

Preferably, the MarR family polypeptide is MarR. Other preferred MarRfamily polypeptides include: EmrR, Ec17kD, and MexR.

In a further embodiment, the MarR family polypeptide has a winged-helixstructure, such as the three dimensional structure of MarR.

FIG. 1 shows a sequence alignment of MarR with representative MarRfamily polypeptides. The MarR secondary structure elements wereidentified in its crystal structure and are illustrated in FIG. 1 (e.g.,as tubes for α-helices (α) and arrows for β-sheets (β) and the singlewing region (W1)). The numbering in FIG. 1 is according to the MarRprimary sequence. The MarR family polypeptides used for the alignmentwere from the following organisms: MarR, E. coli; MprA (EmrR), E. coli;MexR, Pseudomonas aeruginosa; YS87, Mycobacterium tuberculosis; SlyA,Salmonella typhimurium; PecS, Erwinia chrysanthemi; CinR, Butyrivibriofibrisolvens.

In a further embodiment, MarR comprises, consists essentially of, orconsists of the polypeptide sequence shown in Sequence Listing SEQ IDNO:1. Other MarR family polypeptides of interest include EmrR, YS87,PecS, CinR, SlyA, Ec17kD, MexR, etc.

In another embodiment, the MarR family polypeptide is found, forexample, in one of the following organisms Escherichia coli, Salmonellatyphimurium, Salmonella enterica, Enterobacter cloacae, Enterobacteraerogenes, Erwinia chrysanthemi, Yrsinia pestis, Yersiniaenterocolitica, Kluyvera cryocrescens, Edwardsiella tarda, Pseudomonasaeruginosa, Vibrio cholera, Xanthomonas axonopodis, Xanthomonascampestris, Ralstonia solanacearum, Burkholderia pseudomallei,Burkholderia cepacia, Vogesella indigofera, Mesorhizobium loti,Agrobacterium tumefaciens, Sinorhizobium meliloti, Brucella melitensis,Caulobacter crescentus, Bacillus anthracis, Bacillus subtilis, Bacillushalodurans, Listeria monocytogenes, Listeria innocua, Listeriawelshimeri, Staphylococcus sciuri, Streptococcus criceti, Streptococcuspneumoniae, Clostridium perfringens, Clostridium difficile, Streptomycescoelicolor, Streptomyces avermitilis, Mycobacterium tuberculosis,Mycobacterium leprae, Corynebacterium glutamicum, Thermotoga maritima,Methanosarcina acetivorans, Methanosarcina mazei, and Sulfolobussolfataricus.

In another embodiment, the MarR family polypeptide is from an organismbelonging to one of the following biological classifications:Enterobacteriaceae, Enterobacter, Yersinia, Kluyvera, Edwardsiella,Xanthomonas group, Xanthomonadales, Pseudomonaceae/Moraxellaceae group,Pseudomonadaceae, Vibrionaceae group, Burkholderia/Oxalobacter/Ralstoniagroup, Ralstonia group, Burkholderia group, Neisseriaceae, Vogesella,Rhizobiaceae group, Phyllobacteriaceae, Mesorhizobium, Rhizobiaceae,Sinorhizobium, Brucellaceae, Brucella, Caulobacter group, Firmicutes,Bacillus/Clostridium group, Bacilli, Bacillales, Bacillus, Bacillaceae,Bacillus cereus group, Listeria, Listeriaceae, Staphylococcaceae,Staphylococcus, Streptococcus, Lactobacillales, Streptococcaceae,Clostridium, Clostridiaceae, Clostridiales, Clostridia, Actinomycetales,Actinobacteria, Actinobacteridae, Streptomyces, Streptomycineae,Streptomycetaceae, Corynebacterineae, Mycobacterium, Mycobacteriaceae,Corynebacteriaceae, Corynebacterium, Nostocales, Nostocaceae, Nostoc,Thermotogae, Thermotogales, Thermotogaceae; Thermotoga, Methanosarcina,Euryarchaeota, Methanococci; Methanosarcinales, Methanosarcinaceae,Crenarchaeota, Thermoprotei; Sulfolobales, Sulfolobaceae, Sulfolobus,Proteobacteria, Pectobacterium, Cyanobacteria, or Archaea.

In one embodiment, the MarR family polypeptides of the invention arenaturally occurring. In another embodiment, the subject crystalstructures can be generated using non-naturally occurring forms of MarRfamily polypeptides, e.g. mutants or synthetic forms of MarR familypolypeptides not found in nature.

In one embodiment, the MarR family polypeptide comprises one or moreconservative mutations as compared to the wild type protein for theparticular MarR family polypeptide. The term “MarR family polypeptide”also includes fragments of MarR family polypeptides which minimallyretain at least a portion of the tertiary structure of the MarR familyprotein.

MarR family member polypeptide sequences are “structurally related” toone or more known MarR family members, preferably to MarR. Thisstructural relatedness is shown by sequence similarity between two MarRfamily polypeptide sequences or between two MarR family nucleotidesequences. Sequence similarity can be shown, e.g., by optimally aligningMarR family member sequences using an alignment program for purposes ofcomparison and comparing corresponding positions. To determine thedegree of similarity between sequences, they will be aligned for optimalcomparison purposes (e.g., gaps may be introduced in the sequence of oneprotein for nucleic acid molecule for optimal alignment with the otherprotein or nucleic acid molecules). The amino acid residues or bases andcorresponding amino acid positions or bases are then compared. When aposition in one sequence is occupied by the same amino acid residue orby the same base as the corresponding position in the other sequence,then the molecules are identical at that position. If amino acidresidues are not identical, they may be similar. As used herein, anamino acid residue is “similar” to another amino acid residue if the twoamino acid residues are members of the same family of residues havingsimilar side chains. Families of amino acid residues having similar sidechains have been defined in the art (see, for example, Altschul et al.1990. J. Mol. Biol. 215:403) including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan). The degree (percentage) of identity orsimilarity between sequences, therefore, can be calculated as a functionof the number of identical or similar positions shared by two sequences(i.e., % homology=# of identical or similar positions/total # ofpositions×100). Alignment strategies are well known in the art; see, forexample, Altschul et al. supra for optimal sequence alignment.

MarR family polypeptides share some amino acid sequence similarity withMarR. The nucleic acid and amino acid sequences of MarR as well as otherMarR family polypeptides are available in the art. For example, thenucleic acid and amino acid sequence of MarR can be found, e.g., onGeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol.175:1484, or in SEQ ID NO:1).

The nucleic acid and protein sequences of MarR can be used as “querysequences” to perform a search against databases (e.g., either public orprivate) to, for example, identify other MarR family members havingrelated sequences. Such searches can be performed, e.g., using theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J.Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed withthe NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to MarR family nucleic acid molecules. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to MarR proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

MarR family members can also be identified as being similar based ontheir ability to specifically hybridize to the complement of nucleicacid sequences specifying MarR. Such stringent conditions are known tothose skilled in the art and can be found e.g., in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Conditions for hybridizations are largely dependent on the meltingtemperature Tm that is observed for half of the molecules of asubstantially pure population of a double-stranded nucleic acid. Tm isthe temperature in ° C. at which half the molecules of a given sequenceare melted or single-stranded. For nucleic acids of sequence 11 to 23bases, the Tm can be estimated in degrees C. as 2(number of A+Tresidues)+4(number of C+G residues). Hybridization or annealing ofnucleic acid molecules should be conducted at a temperature lower thanthe Tm, e.g., 15° C., 20° C., 25° C. or 30° C. lower than the Tm. Theeffect of salt concentration (in M of NaCl) can also be calculated, seefor example, Brown, A., “Hybridization” pp. 503-506, in The Encyclopediaof Molec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).

Preferably, the nucleic acid sequence of a MarR family member identifiedin this way is at least about 10%, 20%, more preferably at least about30%, more preferably at least about 40% identical and most preferably atleast about 50%, or 60% identical or more with a MarR nucleotidesequence. Preferably, MarR family members have an amino acid sequence atleast about 20%, more preferably at least about 30%, more preferably atleast about 40% identical and most preferably at least about 50%, or 60%or more identical with a MarR amino acid sequence. However, it will beunderstood that the level of sequence similarity among microbialregulators of gene transcription, even though members of the samefamily, is not necessarily high. This is particularly true in the caseof divergent genomes where the level of sequence identity may be low,e.g., less than 20% (e.g., B. burgdorferi as compared e.g., to B.subtilis). For example, the level of amino acid sequence homologybetween MarR and PecS is about 31% and the level of amino acid sequencehomology between MarR and PapX is about 28% when determined as describedabove. Accordingly, structural similarity among MarR family members canalso be determined based on “three-dimensional correspondence” of aminoacid residues. As used herein, the language “three-dimensionalcorrespondence” is meant to includes residues which spatiallycorrespond, e.g., are in the same functional position of a MarR familyprotein member as determined, e.g., by x-ray crystallography, but whichmay not correspond when aligned using a linear alignment program. Thelanguage “three-dimensional correspondence” also includes residues whichperform the same function, e.g., bind to DNA or bind the same cofactor,as determined, e.g., by mutational analysis.

Preferred MarR family polypeptides include: MarR, EmrR, Ec17kD, MexR,PapX, SlyA, Hpr, PecS, Hpr, MprA, (EmrR), as well as the other peptideslisted in the chart above or known in the art. In a more preferredembodiment, a MarR family polypeptide is selected from the groupconsisting of MarR, EmrR, Ec17kD, and MexR. In a particularly preferredembodiment, a MarR family polypeptide is MarR.

In addition to sharing structural similarity, MarR family members have aMarR family polypeptide activity, i.e., they bind to DNA and regulatetranscription. Some MarR family members positively regulatetranscription (e.g., SlyA, BadR, NhhD, or MexR), while others negativelyregulate transcription (e.g., MarR). While all MarR family members bindto DNA and regulate transcription, the different loci controlled by eachfamily member regulate different processes in microbes. For example,MarR family polypeptides can control the expression of microbial lociinvolved in: regulation of antibiotic resistance [e.g., MarR (Cohen etal. 1993. J. Bacteriol. 175:1484), EmrR (Lomovskaya and Lewis. 1992.Proc. Natl. Acad. Sci. 89:8938), and Ecl7 kD (Sulavik et al. 1995. Mol.Med. 1:436), and MexR (Poole et al. 1996. Antimicrob. Agents. Chemother.40:2021)1, regulation of tissue-specific adhesive properties [e.g., PapX(Marklund et al., 1992. Mol. Microbiol. 6:2225)], regulation ofexpression of a cryptic hemolysin [e.g., SlyA (Ludwig et al. 1995249:4740)], regulation of protease production [e.g., Hpr from B.subtilis (Perago and Hoch. 1988. J. Bacteriol. 170:2560) and PecS fromErwinia chrysanthemi (Reverchon et al., 1994. Mol. Microbiol. 11:1127)]and regulation of sporulation [e.g., Hpr (Perego and Hoch. 1988. J.Bacteriol. 170:2560)], regulation of the breakdown of plant materials[e.g., CinR (Dalymple and Swadling 1997 Microbiology)] sensing ofphenolic compounds [(e.g., Sulvik et al. 1995. Mol. Med. 1:436], andrepress marRAB expression when introduced into E. coli [e.g., Ec17 kd(Markhmd et al. 1992. Mol. Microbiol. 6:2225) and MprA (EmrR) (delCastillo et al., 1991. J. Bacteriol. 173:3924)]. The activity of MarRfamily polypeptides is antagonized by salicylate (Lomovskaya et al.,1995. J. Bacteriol. 177:2328; Sulavik et al. 1995. Mol. Med. 1:436).

Preferred MarR family polypeptide activities include regulation ofmultiple drug resistance and/or regulation of virulence.

In addition to full length MarR family polypeptide fragments MarR familypolypeptide which are useful in making crystals are also within thescope of the invention. Accordingly, MarR family polypeptides for use inthe instant invention can be full length MarR family member proteins orfragments thereof. Thus, a MarR family polypeptide can comprise, consistessentially of, or consist of an amino acid sequence derived from thefull length amino acid sequence of a MarR family member. For example, inone embodiment, a polypeptide comprising a MarR family polypeptide DNAinteracting domain or a polypeptide comprising a MarR family memberprotein interacting domain can be used.

In addition, naturally or non-naturally occurring variants of thesepolypeptides and nucleic acid molecules which retain the same functionalactivity, e.g., the ability to bind to DNA and regulate transcription.Such variants can be made, e.g., by mutation using techniques which areknown in the art. Alternatively, variants can be chemically synthesized.

For example, it will be understood that the MarR family polypeptidesdescribed herein, are also meant to include equivalents thereof. Forinstance, mutant forms of MarR family polypeptides which arefunctionally equivalent, (e.g., have the ability to bind to DNA and toregulate transcription from an operon) can be made using techniqueswhich are well known in the art. Mutations can include, e.g., at leastone of a discrete point mutation which can give rise to a substitution,or by at least one deletion or insertion. For example, randommutagenesis can be used. Mutations can be made by random mutagenesis orusing cassette mutagenesis. For the former, the entire coding region ofa molecule is mutagenized by one of several methods (chemical, PCR,doped oligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. In thelatter, discrete regions of a protein, corresponding either to definedstructural or functional determinants (e.g., the first or second helixof a helix-turn-helix domain) are subjected to saturating or semi-randommutagenesis and these mutagenized cassettes are re-introduced into thecontext of the otherwise wild type allele. In one embodiment, PCRmutagenesis can be used. For example, Megaprimer PCR can be used (O. H.Landt, Gene 96:125-128).

In addition, other portions of the above described polypeptides suitablefor use in the claimed assays, such as those which retain their function(e.g., the ability to bind to DNA, to regulate transcription from anoperon) or those which are critical for binding to regulatory molecules(such as compounds) can be easily determined by one of ordinary skill inthe art, e.g., using standard truncation or mutagenesis techniques andused in the instant assays. Exemplary techniques are described byGallegos et al. (1996. J. Bacteriol. 178:6427).

It shall be understood that the instant invention also pertains toisolated MarR family member polypeptides, portions thereof, and thenucleic acid molecules encoding them, including naturally occurring andmutant forms.

Preparation of MarR Family Polypeptides

Preferred MarR family polypeptides for use in the instant invention aresynthesized, isolated or recombinant polypeptides. In one embodiment,MarR family polypeptides can be made from nucleic acid molecules.Nucleic acid molecules encoding MarR family polypeptides can be used toproduce MarR family polypeptides for use in the instant assays. Forexample, nucleic acid molecules encoding a MarR family polypeptide canbe isolated (e.g., isolated from the sequences which naturally flank itin the genome and from cellular components) and can be used to produce athe MarR family polypeptide is expressed in a form that is secreted fromcells, the medium can be collected. Alternatively, if the MarR familypolypeptide is expressed in a form that is retained by cells, the hostcells can be lysed to release the MarR family polypeptide. Such spentmedium or cell lysate can be used to concentrate and purify the MarRfamily polypeptide. For example, the medium or lysate can be passed overa column, e.g., a column to which antibodies specific for the MarRfamily member polypeptide have been bound. Alternatively, suchantibodies can be specific for a non-MarR family member polypeptidewhich has been fused to the MarR family polypeptide (e.g., as a tag) tofacilitate purification of the MarR family member polypeptide. Othermeans of purifying MarR family member polypeptides are known in the art.

Architecture of the MarR-Salicylate Co-Crystal Structure

The term “three dimensional structure” includes both pictorialrepresentations of MarR family polypeptides (e.g., such as those shownfor MarR with salicylate and MarR without salicylate in the Figures) aswell as atomic coordinates (e.g., such as those given in FIG. 7 forMarR-salicylate cocrystal, or in FIG. 8 for MarR without salicylate) andother renditions of the shape, size, or symmetry of a MarR familypolypeptide of interest. In a further embodiment, the three dimensionalstructure of the crystallized MarR family polypeptide is determined to aresolution of 5 Å or better, 3 Å or better, 2.5 Å or better, or,advantageously, 2.3 Å or better. The three dimensional structure ofMarR, a MarR family polypeptide, is described in greater detail below.

MarR consists of a dimer with approximate overall dimensions of 50×55×45Å (corresponding to the width, height and depth of the molecule in theorientation shown in FIG. 2). There is one monomer in the asymmetricunit of the crystal with the dimer composed of subunits related by acrystallographic two-fold rotation. The dimeric structure is consistentwith the results of earlier in vitro experiments suggesting that MarRbinds the mar operator (marO) as a dimer (Martin, R. G. et al. supra(1996); Martin, R. G. & Rosner, J. L. Proc. Natl. Acad. Sci. U.S.A. 92,5456-5460 (1995)). Another family member, MprA (EmrR) (FIG. 1) is alsobelieved to function as a dimer (Brooun, A., et al. J. Bact. 181,5131-5133 (1999)).

Each MarR subunit is an α/β protein with approximate dimensions of35×25×60 Å and can be divided into two domains as shown in FIG. 2. FIG.2 is a ribbon representation of the co-crystal structure of the MarRdimer viewed with the subunit 2-fold axis near vertical. The secondarystructure elements of one subunit are colored according to the schemeused in FIG. 1. The N- and C-terminal regions are closely juxtaposed andintertwine with the equivalent regions of the second subunit to form adomain that holds the subunits together (FIG. 5). This N-/C-terminaldomain is linked MarR family polypeptide. In one embodiment, a nucleicacid molecule which has been (1) amplified in vitro by, for example,polymerase chain reaction (PCR); (2) recombinantly produced by cloning,or (3) purified, as by cleavage and gel separation; or (4) synthesizedby, for example, chemical synthesis can be used to produce MarR familypolypeptides. As used herein, the term “nucleic acid” refers topolynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA).

Nucleic acid molecules specifying MarR family polypeptides can be placedin a vector. The term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid molecule to which it has beenlinked. The term “expression vector” includes any vector, (e.g., aplasmid, cosmid or phage chromosome) containing a gene construct in aform suitable for expression by a cell (e.g., linked to a promoter). Inthe present specification, “plasmid” and “vector” are usedinterchangeably, as a plasmid is a commonly used form of vector.Moreover, the invention is intended to include other vectors which serveequivalent functions.

Exemplary expression vectors for expression of a gene encoding a MarRfamily polypeptide and capable of replication in a bacterium, such abacterium from a genus selected from the group consisting ofEscherichia, Bacillus, Streptomyces, Streptococcus, or in a cell of asimple eukaryotic fungus such as a Saccharomyces or, Pichia, or in acell of a eukaryotic organism such as an insect, a bird, a mammal, or aplant, are known in the art. Such vectors may carry functionalreplication-specifying sequences (replicons) both for a host forexpression, for example a Streptomyces, and for a host, for example, E.coli, for genetic manipulations and vector construction. See e.g. U.S.Pat. No. 4,745,056. Suitable vectors for a variety of organisms aredescribed in Ausubel, F. et al., Short Protocols in Molecular Biology,Wiley, New York (1995), and for example, for Pichia, can be obtainedfrom Invitrogen (Carlsbad, Calif.).

Useful expression control sequences, include, for example, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. A useful translational enhancer sequenceis described in U.S. Pat. No. 4,820,639.

It should be understood that the design of the expression vector maydepend on such factors as the choice of the host cell to be transformedand/or the type of protein desired to be expressed.

“Transcriptional regulatory sequence” is a generic term to refer to DNAsequences, such as initiation signals, enhancers, operators, andpromoters, which induce or control transcription of nucleic acidsequences with which they are operably linked. It will also beunderstood that a recombinant gene encoding a MarR family polypeptidecan be under the control of transcriptional regulatory sequences whichare the same or which are different from those sequences which controltranscription of the naturally-occurring MarR family gene. Exemplaryregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences, that control the expression of a DNA sequence whenoperatively linked to it, may be used in these vectors to express DNAsequences encoding the MarR family proteins of this invention.

Appropriate vectors are widely available commercially and it is withinthe knowledge and discretion of one of ordinary skill in the art tochoose a vector which is appropriate for use with a given microbialcell. The sequences encoding MarR family polypeptides can be introducedinto a cell on a self-replicating vector or may be introduced into thechromosome of a microbe using homologous recombination or by aninsertion element such as a transposon.

Such vectors can be introduced into cells using standard techniques,e.g., transformation or transfection. The terms “transformation” and“transfection” mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient or “host” cell. The term“transduction” means transfer of a nucleic acid sequence, preferablyDNA, from a donor to a recipient cell, by means of infection with avirus previously grown in the donor, preferably a bacteriophage. Nucleicacids can also be introduced into microbial cells by transformationusing calcium chloride or electroporation.

“Cells,” “host cells,” “recipient cells, are terms used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. In preferred embodiments, cells used to express MarR familypolypeptides for purification, e.g., host cells, comprise a mutationwhich renders any endogenous MarR family polypeptide nonfunctional orcauses the endogenous polypeptide to not be expressed. In otherembodiments, mutations may also be made in other related genes of thehost cell, such that there will be no interference from the endogenoushost loci.

Purification of a MarR family polypeptides, e.g., recombinantlyexpressed polypeptides, can be accomplished using techniques known inthe art. For example, if to the remainder of the protein by two longantiparallel helices in each subunit. These helices lead to a globulardomain that is likely to be responsible for DNA binding (see below).Although the globular DNA-binding domains of the dimer are adjacent toone another, they make minimal contact with each other and are situatedto function independently. The overall organization of the N-/C-terminaldomain and the two DNA-binding domains results in the formation of anapproximately 6 {acute over (Å)} wide channel through the center of thedimer (FIGS. 3 and 4). The electrostatic surface potential (FIG. 3) isconsistent with the putative DNA-binding regions being stronglyelectropositive, as observed in other such winged-helix DNA-bindingproteins (Gajiwala, K. S. & Burley, S. K. Curr. Opin. Str. Biol. 10,110-116 (2000)).

Genetic and biochemical data have previously identified the N-terminusof MarR to be important for mediating protein-protein contacts betweenrepressor subunits and have demonstrated that the C-terminus isimportant for protein function (Alekshun, M. N., et al. Mol. Microbiol.35, 1394-404 (2000); Linde, H. J. et al. supra). The present structureshows that α-helices in the N- and C-terminal regions of each monomerfold around and interdigitate with those of the other subunit to form awell-packed hydrophobic core (FIG. 5) burying a surface area of 3,570 Å²(the total buried surface area for the whole dimer is 3,700 Å²). Thedimer is further stabilized in this region by several intermolecularhydrogen bonds, notably that between the c-amino group of Lys 24 and themain chain carbonyl oxygen of Pro 144′ in the C-terminus of the secondsubunit and that between the main chain carbonyl oxygen of Glu 10 andthe side chain amino group of Lys 140′.

While the DNA-binding lobe of each subunit also forms a well-packedhydrophobic core, the only interactions between these lobes of the twosubunits are salt bridges formed between Asp 67 and Arg 73′ and thereciprocal pair (FIG. 6). These salt bridges stabilize the relationshipbetween the two lobes of the dimer in the crystal form of the proteinbut if disrupted by other interactions, such as might occur during thebinding of MarR to marO, the two lobes would be able to actindependently. Relative movement of the lobes would require distortionof the helices that link them to the N-/C-terminal domain. The longlinker helix region encompassing residues 103-126 (α5/α5′) (FIG. 2)appears poorly ordered in the region of Gly 116, as is the loop(residues 128-131) that connects this helix to the C-terminal helix(α6/α6′) (FIGS. 1 and 2). It is possible that flexibility at these sitesin MarR helps to accommodate relative shifts of the two lobes of thedimer that might occur on binding to DNA.

Architecture of the MarR Crystal Structure

The MarR without salicylate structure is a dimer (FIG. 9) and bothsubunits of the dimer are in the asymmetric unit. These individualsubunits are joined by extensive protein-protein interactions mediatedby amino acids within both the N- and C-termini of the monomers (FIG.9). Like the MarR-salicylate structure, MarR without salicylate is anα/β protein. The MarR without salicylate structure is, however,conformationally different from the salicylate bound protein in that thecaliper created by the dimer is more closed in the form of the proteinwithout salicylate. Thus, the channel through the center of the dimerhas been lost.

The overall architecture of the MarR without salicylate structure iscomparable to that of the salicylate bound protein. The presumed DNAbinding lobes or domains are linked to the remainder of the protein bytwo long α-helices. The positioning of the two DNA binding lobes in theMarR without salicylate structure is fixed by hydrogen bonds between thetwo lobes. This arrangement is believed to be mediated by interactionsbetween Asp 67 and Arg 77′. In addition, Asp 26 is involved in hydrogenbonds with the side chains of Lys 44 and Lys 25. Together, the presumedrecognition helices within the DNA binds lobes overlap by approximatelyone helical turn.

The DNA Binding Domain

Previous studies have shown the region spanning amino acids 61-121 inMarR to be required for its DNA binding activity (Alekshun, M. N et al.,supra, (2000)). In the crystal structure, amino acids 55-100[β1-α3-α4-β2-W1 (wing)-β3] adopt the winged-helix fold (Clark, K. L. etal. Nature 364, 412-420 (1993)). The overall topology [H1 (α2)-S1(β1)-H2 (α3)-H3 (α4, recognition helix)-S2 (β2)-W1-S3 (β3)] of thisregion is similar to other winged-helix DNA binding proteins (theterminology applied for these and subsequent structural elements isaccording to Gajiwala and Burley, supra (2000)) except that a thirdstrand of sheet present in most members of the group appears to berepresented in this MarR structure by an interaction with Ile 55 (β1)(FIG. 1). The presence of this single residue as the third component inthe sheet interaction is very similar to that observed in OmpR(Martinez-Hackert, E. & Stock, A. M. Structure 5, 109-124 (1997)), awinged helix protein, where Leu 180 interacts with the two strands ofthe antiparallel sheet that forms part of the “wing” in thistranscription factor.

Within the winged-helix family of DNA-binding proteins, there aremultiple modes of DNA binding. Members such as HNF-3γ use therecognition helix (H3) of the motif as the primary determinant forDNA-protein interactions in the major groove, and a wing region(s) (W1)to form minor groove or phosphodiester backbone nucleoprotein contacts(Clark, K. L. et al. supra (1993)). Others, such as hRFX1, use W1 tointeract with the major groove and the H3 helix makes only a singleminor groove contact (Gajiwala, K. S. et al. Nature 403, 916-921(2000)). The juxtaposition of the DNA-binding lobes in the presentstructure does not allow for modeling of the whole dimer onto a B-DNArepresentation of the operator. However, since mutations in both α4 (H3)and W1 affect the DNA binding activity of MarR it is expected that aminoacids from each of these regions would contribute to the DNA bindingactivity of the protein. For example, mutations in a4, including an R73Cchange, abolish MarR DNA binding activity in whole cells and in vitro(Alekshun, M. N et al., supra, (2000)). In the present crystalstructure, it is the side chain of Arg 73 that is hydrogen bonded to Asp67′ of the other subunit, an interaction that stabilizes the relativeorientation of the two DNA-binding lobes (FIG. 6). Also, an R94Cmutation at the tip of W1 is inactive in a whole cell assay while a G95S“superrepressor” mutation increases the DNA binding activity of MarR30-fold in vitro (Alekshun, M. N et al., supra, (2000); Alekshun, M. N.& Levy, S. B. J. Bact. 181, 3303-3306 (1999)). In the absence ofprotein-DNA co-crystal structures, the precise mechanism by which thesemutations affect the DNA binding activity of the protein is uncertain.

Footprinting experiments have suggested that MarR binds as a dimer attwo separate but very similar sites in marO, the protein protects ˜21-bpof DNA on both strands at a single site, and does not bend its target(Martin, R. G. et al., supra (1996); Martin, R. G. et al. Proc. Natl.Acad. Sci. U.S.A. 92, 5456-5460 (1995)). Each MarR binding site iscomposed of two half-sites whose organization is such that they are ondifferent faces of the DNA double helix (Alekshun, M. N. et al. Mol.Microbiol. 35, 1394-404 (2000)), an arrangement that is very similar tothe hRFX1 binding site (Gajiwala, K. S. et al. Nature 403, 916-921(2000)). For MarR to bind as a dimer, with each winged-helix DNA bindingdomain contacting one half-site on B-DNA, geometric constraints suggestonly a few possible modes of binding. One scenario, involving thebinding of a single dimer to one MarR binding site, would requirereorientation of the DNA binding lobes so that each could reach onehalf-site. This would be analogous to the binding of an E2F-DPheterodimer (a eukaryotic transcription factor in which each subunitalso has a winged-helix DNA binding domain) to its cognate binding site(Zheng, N. et al. Genes Dev. 13, 666-74. (1999)). A second scenariowould involve the binding of two dimers, on opposite faces of the doublehelix, to a single MarR binding site. This model would be analogous tothe binding of DtxR (a bacterial protein with a winged-helix DNA bindingdomain) to its target, although in DtxR the half-sites are on the sameface of the DNA helix (Pohl, E. et al. J. Biol. Chem. 273, 22420-22427(1998); White, A. et al. Nature 394, 502-506 (1998)).

The term “appropriate conditions” include those conditions which resultin the formation of a crystal which can by analyzed to a resolution of5.0 Å or less. The crystals may be formed using suitable art recognizedtechniques, such as hanging droplet vapor diffusion. In one embodiment,the temperature of crystallization of the MarR family polypeptide isfrom about 1° C. to about 30° C., from about 10° C. to about 25° C.,from about 15° C. to about 20° C., or abut 17° C. In a furtherembodiment, the conditions are selected such that crystals of said MarRfamily polypeptide grow within an acceptable time and reach dimensionswhich are suitable for structural determination, e.g., by using X-raydiffraction. In one embodiment, the acceptable time is 8 weeks or less,6 weeks or less, 4 weeks or less, or 3 weeks or less. In an embodiment,the dimensions of the crystal are approximately 0.1 mm or greater perside, 0.2 mm or greater per said, or approximately 0.3 mm per side orgreater.

In a further embodiment, the appropriate conditions include acocrystallization agent which interacts with the protein such that thethree dimensional structure of the protein can be determined.

The term “cocrystallization agent” includes substances which can becrystallized with the MarR family polypeptide such that the threedimensional structure can be determined. In an embodiment, thecocrystallization agent is a MarR family polypeptide modulator. The term“MarR family polypeptide modulator” includes compounds which interactwith MarR, either to inhibit or enhance the activity of MarR, such thatthey alter its activity in its non-crystallized form. In one embodiment,the MarR family polypeptide modulator is a MarR inhibitor (e.g.,salicylate, plombagin, or DNP). In an embodiment, the concentration ofthe salicylate is about 100 mM or less, 150 mM or less, 200 mM or less,or 250 mM or less.

The crystal structure or MarR has been solved using crystals grown inthe presence and in the absence of high concentrations (250 mM) ofsodium salicylate. This agent, at millimolar concentrations, is known toinhibit MarR activity both in vitro and in whole cells (Alekshun, M. N.supra (1999)). It is routinely used as a model inhibitor of MarR toinduce MarA expression in E. coli and S. typhimurium (Cohen, S. P. etal. J. Bact. 175, 7856-7862 (1993); Sulavik, M. C. et al. J. Bact. 179,1857-1866 (1997)) and thus, to confer a Mar phenotype (Alekshun, M. N.supra (1999)). In one example, salicylate was included in the currentcrystal growth conditions to provide stable crystals. In anotherexample, the crystal structure of MarR was determined using MarR withoutsalicylate.

Electron density that is consistent with bound salicylate is apparent attwo sites on each subunit in the present structure (FIG. 6). These sitesare on the surface of the molecule on either side of the proposedDNA-binding helix α4 (H3). In one site (SAL-A), the salicylate hydroxylis hydrogen bonded to the hydroxyl side chain of Thr 72 in the α4 (H3)helix and the salicylate carboxylate hydrogen bonds to the guanidiniumgroup of Arg 86. In the other site (SAL-B), the salicylate hydroxylhydrogen bonds to the backbone carbonyl of Ala 70 and its carboxylhydrogen bonds to Arg 77. In each of these sites, the salicylate ringsits over a hydrophobic side chain in the pocket; Pro 57 in SAL-A andMet 74 in SAL-B and other surface hydrophobes are also located laterallywithin 3.5 {acute over (Å)} of the unsubstituted side of the ring.Although SAL-B is solvent exposed, SAL-A packs in the crystal with Val96 of a symmetry mate situated 3.6 Å above the salicylate ring andadjacent to the SAL-A site of this symmetry mate. Since both SAL-A andSAL-B are close to the DNA binding helix, they may be positioned toinfluence DNA binding.

The crystal structure of MarR was solved by multiwavelength anomalousdispersion methods using protein containing selenomethionine.Diffraction data were collected to 2.3 Å from crystals of both selenoand native protein.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXEMPLIFICATION OF THE INVENTION Example 1 Crystallization of MarR withSalicylate

Protein Production and Purification

Native and selenomethionine (Se-Met) containing MarR was prepared fromE. coli BL21(DE3) (Novagen) bearing pMarR-WT, a wild type MarRexpression vector that has been previously described (Alekshun, M. N. &Levy, S. B. J. Bact. 181, 4669-4672 (1999)). Native MarR was produced inwhole cells according to previous methods (Alekshun, M. N. & Levy, S. B.J. Bact. 181, 4669-4672 (1999)). Se-Met MarR was produced by diluting anovernight culture of E. coli BL21(DE3)+pMarR-WT 1:1000 in M9 mediumsupplemented with 2 mM MgSO₄, 0.2% glucose, 0.1 mM CaCl₂, 0.00005%thiamine, 0.04 mg ml⁻¹ each of the following amino acids phenylalanine,leucine, isoleucine, valine, serine, threonine, tyrosine, histidine,lysine, aspartic acid, glutamic acid, tryptophan, and tryptophan, andkanamycin (Miller, J. H. In Experiments in Molecular Genetics. (ColdSpring Harbor Laboratories, Cold Spring Harbor, N.Y.; 1972). Thisculture was grown at 37° C. to an OD600≈0.6 and 100 mg each of aminoacids threonine, lysine-hydrochloride, phenylalanine, 50 mg each ofamino acids leucine, isoleucine, and valine (single letterabbreviations), and 60 mg L-(+)-selenomethionine (Sigma) were thenadded. The culture was grown for 15 min at 37° C.; IPTG was subsequentlyadded to a final concentration of 1 mM and protein production wasallowed to proceed for 14.5 hr at 37° C. Cell pellets were collected andprocessed as previously described (Alekshun, M. N. & Levy, S. B. J.Bact. 181, 4669-4672 (1999)).

Frozen cell pellets containing native or Se-Met MarR were resuspended in100 mM sodium phosphate buffer (pH 7.4) containing a bacterial proteaseinhibitor cocktail (Sigma) and sonicated on ice. All buffers contained 2mM DTT when Se-Met MarR was prepared. Insoluble matter was removed bycentrifugation at 4° C. at 30,000×g for 40 min. The supernatant waspassed over prepacked 5 ml SP-sepharose HiTrap columns (AmershamPharmacia Biotech) previously equilibrated with 10 mM sodium phosphatebuffer (pH 7.4). The column was washed with 50 ml of 10 mM sodiumphosphate buffer (pH 7.4) and the pure proteins were eluted with alinear gradient (0-0.5 M) of NaCl in 10 mM sodium phosphate buffer (pH7.4). Protein containing fractions were dialyzed vs. 10 mM HEPES (pH7.4), 200 mM NaCl, and 1 mM DTT, or 2 mM DTT in the case of Se-Met MarR,and the protein in these samples was judged to be greater than 99% purevia SDS-PAGE and electrospray ionization mass spectrophotometry. Thelatter also demonstrated that more than 95% of the three methionineresidues in Se-Met MarR were substituted with selenomethionine.

Crystallization:

MarR crystals were originally grown in 18% PEG MME 5000, 200 mM ammoniumsulfate, 100 mM citrate buffer (pH 5.6) but showed anisotropic disorderin the diffraction data that made them unsuitable for structuredetermination. To stabilize the protein, the citrate was substituted bythe known inhibitor salicylate. Crystals of the MarR-salicylate complexwere grown at 17° C. by hanging droplet vapor diffusion. 6 p. 1 of a11.4 mg ml⁻¹ protein solution in 200 mM NaCl, 20 mM HEPES (pH 7.4), and10 mM DTT were added to 2 μl of reservoir buffer (18% PEG MME 5000, 50mM ammonium sulfate, 250 mM sodium salicylate, 10 mM DTT, and 15%glycerol, pH 5.5), and 0.8 μl 15% heptanetriol. The droplets wereequilibrated with 1 ml of reservoir buffer. Crystals grew within 1 weekreaching dimensions of approximately 0.3 mm per side.

X-Ray Data Collection, Structure Determination, and Refinement:

Diffraction data were collected at the Brookhaven National SynchrotronLight Source, beamline X8C. Crystals were flash frozen in mother liquorat the beam line before data collection. All data were processed andreduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4 Proceedings.56-62 (Daresbury Laboratory, Warrington, UK, 1993). The space group ofthe MarR-salicylate co-crystals was determined to be I4₁22 with onemolecule in the asymmetric unit and with unit cell dimensions ofa=b=62.0 Å, c=132.9 Å, α=β=β=90° for both the native and theselenoprotein. Data were collected on the selenoprotein crystals atthree wavelengths to enable MAD phasing. Phases were determined from theMAD data using the program SOLVE (Terwilliger, T. C. & Berendzen, J.Acta. Crystallogr. D. 55, 849-861 (1999)). This showed two seleniumsites per asymmetric unit, with the third selenomethionine, at theN-terminus, apparently disordered. Maps were solvent-flattened using theprogram DM and the model was built into density using the program O(Collaborative Computational Project, Number 4. Acta Crystallogr. D. 50,760-763 (1994); Jones, T. A. et al. Acta Crystallogr. A 47, 110-119(1991)). Model and refinement parameters for salicylate were obtainedfrom the Hetero Compound Information Center (Kleywegt, G. J. & Jones, T.A. Acta Crystallogr. D. 54, 1119-1131 (1998)). Model refinement wasperformed using CNS and cycles of rebuilding and refinement continued togive the final model (Brunger, A. T. et al. Acta. Crystallogr. D. 54,905-921 (1998)). Model quality was assessed by sa-omit, Fo-Fc, mapsgenerated over the whole molecule omitting no more than 7% of thestructure at a time. The model extends from residue 6 to the C-terminusat residue 144. In common with several other transcription factors (e.g.TetR, (1A6I), ArgR (1B4B) and TreR (1BYK)), MarR shows relatively highthermal mobility throughout the structure, as reflected by theB-factors. Certain regions appear to be particularly mobile, includingthe extended structure at the N-terminus, the tip of the “wing”(residues 91-94), parts of the α5 helix, especially around Gly 116 andthe connecting loop (128-131) between the α5 and the C-terminal α6helix. Consistent with the high B-factors, the molecule shows fewwell-ordered solvent molecules. PROCHECK reports overall g-factors of0.25 (dihedrals) and 0.55 (main chain covalent forces) and shows that91% of the residues fall within the most favored region of theRamachandran plot, with only residue Ala 53 in a disallowed region. Thisresidue is located at the start of the loop connecting the α2 and α3helices.

The coordinates of the MarR-salicylate cocrystal are shown in FIG. 7.Table 1 shows data collection, phasing, and refinement statistics forthe MarR-salicylate co-crystal.

TABLE 1 Data set Native Se-met edge Se-met peak Se-met remote Wavelength(Å) 1.072 0.9795 0.9793 0.9500 Resolution range (Å) 50-2.3 50-2.3 50-2.350-2.3 Measured reflections 56,495 84,173 96,582 87,365 Uniquereflections 6,069 5,534 5,564 5,472 Completeness (%) overall (final 99.5(100)  91.3 (99.8) 91.7 (99.8) 90.4 (99.7) <I/σI> (final shell) 21.1(12.0) 12.2 (7.2)  12.0 (7.0)  12.9 (7.9)  R_(merge)(%) (final shell) 6.0 (20.0)  6.4 (29.7)  5.7 (30.3)  4.9 (25.5) Rano(%) 4.9 5.0 3.5Overall FOM (centric/acentric) 0.59/0.71 Resolution 50-2.3 Rfree 28.7%Rcryst 24.7% Atoms/AU Protein 1078 Salicylate 20 Water 18 Average B (Å²)main chain 49.7 side chain 59.2 salicylate 42.7 water 50.0 R.m.s.deviation Bonds (Å) 0.009 Angles (°) 1.3

Example 2 Crystallization of MarR

MarR was produced and purified as described in Example 1.

Crystallization:

Crystals of MarR were grown by hanging droplet vapor diffusion. 3 μl ofa 10 mg ml⁻¹ 2:1 (mol:mol) DNA-protein solution in 200 mM NaCl, 20 mMHEPES, pH 7.4, 20 mM TRIS-HCl, pH 8.0, and 2 mM MgCl₂ was added to 1 μlof reservoir buffer (23% PEG MME 5000, 100 mM sodium citrate, 200 mMammonium sulfate, 10 mM DTT, 10% glycerol, 5% Isopropanol, pH 5.6), and0.4 μl 15% heptanetriol. The droplets were equilibrated with 0.5 ml ofreservoir buffer.

X-ray data collection, structure determination, and refinement:

Diffraction data were collected at the Brookhaven National SynchrotronLight Source, beamline X8C. Crystals were flash frozen in mother liquorat the beam line before data collection. All data were processed andreduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4 Proceedings.56-62 (Daresbury Laboratory, Warrington, UK, 1993).

The coordinates of the MarR crystal without salicylate are shown in FIG.8. Table 2 shows data collection, phasing, and refinement statistics forthe MarR crystal.

TABLE 2 Space group C222 Unit cell (Å) a = 65.8, b = 137.7, c = 96.4Resolution 50-2.7 Rfree 26.7% Rcryst 23.2% Atoms/AU Protein 2093 Water14 Average B (Å²) main chain 40.0 side chain 48.0 Water 32.6 R.m.s.deviation Bonds (Å) 0.009 Angles (°) 1.3

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference. The entire contents ofAlekshun et al. “The Crystal Structure of MarR a Regulator of MultipleAntibiotic Resistance at 2.3 Å resolution,” Nature Structural Biology8(8) is hereby incorporated herein by reference.

1. A crystallized MarR family polypeptide, wherein said crystallizedMarR family polypeptide is crystallized under appropriate conditionssuch that the three dimensional structure of said MarR familypolypeptide can be determined.
 2. The crystallized MarR familypolypeptide of claim 1, wherein the crystallized MarR family polypeptideis a structural homolog of crystallized MarR.
 3. The crystallized MarRfamily polypeptide of claim 1, wherein said MarR family polypeptide isselected from the group consisting of MarR, SlyA, EmrR (MprA), PapX,PrsX, HpcR, Ec17 kD of Escherichia coli; MarR, SlyA, EmrR of Salmonellatyphimurium; MexR of Pseudomonas aeruginosa; PecS of Erwiniachrysanthemi; BadR of Rhodopseudomonas palustris; OrfE of Burkholderiapseudomallei; YdcH, YhbI, YkmA, YkoM, Orf7, YfiV, YetL, YdgJ, YwoH,YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO, YpoP, YkvE of Bacillussubtilus; Orf7 of Bacillus firmus; Orf145, Orf141 of Staphylococcussciuri; CinR of Butyrivibrio fibrisolvens; Orf158 of Sphingomonasaromaticivorans; NhhD of Rhodococcus rhodochrous; Orf1 of Streptomycespeucetius; 14.7 kD, Rv1404, Rv0737, Rv0042c, Yz08 (15.6 kD) ofMycobacterium tuberculosis; Yz08 (15.6 kD) of Mycobacterium leprae;MTH313 of Methanobacterium thermoautotrophicum; Lrs14 of Sulfolobussolfataricus; CinR of Archaeoglobus fulgidus; PetP of Rhodobactercapsulatus; and SlyA (E293909) of Sinorhizobium meliloti.
 4. Thecrystallized MarR family polypeptide of claim 1, wherein said MarRfamily polypeptide is MarR.
 5. The crystallized MarR family polypeptideof claim 4, wherein the peptide sequence of MarR comprises the aminoacid sequence shown in SEQ ID NO:2.
 6. The crystallized MarR familypolypeptide of claim 4, wherein said three dimensional structure has thespace group of I4₁22.
 7. The crystallized MarR family polypeptide ofclaim 1, wherein said appropriate conditions include the presence of aMarR family polypeptide modulator.
 8. The crystallized MarR familypolypeptide of claim 7, wherein said MarR family polypeptide inhibitoris salicylate.
 9. The crystallized MarR family polypeptide of claim 1,wherein said three dimensional structure is determined to a resolutionof 5 Å or better. 10.-12. (canceled)
 13. The crystallized MarR familypolypeptide of claim 4, wherein said crystallized MarR familypolypeptide has a structure defined by the atomic coordinates given inFIG. 7 or FIG.
 8. 14. (canceled)
 15. The crystallized MarR familypolypeptide of claim 1, wherein said MarR family polypeptide has awinged-helix structure.
 16. A crystallized MarR family polypeptidedefined by the atomic coordinates given in FIG. 7 or FIG.
 8. 17.(canceled)
 18. A method for determining the three dimensional structureof MarR family polypeptide, comprising crystallizing the MarR familypolypeptide under appropriate conditions such that crystals are formed;and analyzing the crystallized MarR family polypeptide, such that thethree dimensional structure of the MarR family polypeptide isdetermined.
 19. The method of claim 18, wherein said crystallized MarRfamily polypeptide is analyzed by x-ray diffraction techniques.
 20. Themethod of claim 18, wherein said MarR family polypeptide is MarR. 21.The method of claim 20, wherein MarR comprises the amino acid sequenceset forth in SEQ ID NO:2.
 22. The method of claim 18, wherein saidappropriate conditions comprise the presence of a MarR familypolypeptide modulator.
 23. The method of claim 22, wherein said MarRfamily polypeptide modulator is salicylate.
 24. The method of claim 18,wherein said three dimensional structure is determined to a resolutionof 5 Å or better. 25.-27. (canceled)
 28. The method of claim 18, whereinsaid MarR family polypeptide is selected from the group consisting of:MarR, SlyA, EmrR (MprA), PapX, PrsX, HpcR, Ec17 kD of Escherichia coli;MarR, SlyA, EmrR of Salmonella typhimurium; MexR of Pseudomonasaeruginosa; PecS of Erwinia chrysanthemi; BadR of Rhodopseudomonaspalustris; OrfE of Burkholderia pseudomallei; YdcH, YhbI, YkmA, YkoM,Orf7, YfiV, YetL, YdgJ, YwoH, YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO,YpoP, YkvE of Bacillus subtilus; Orf7 of Bacillus firmus; Orf145, Orf141of Staphylococcus sciuri; CinR of Butyrivibrio fibrisolvens; Orf158 ofSphingomonas aromaticivorans; NhhD of Rhodococcus rhodochrous; Orf1 ofStreptomyces peucetius; 14.7 kD, Rv1404, Rv0737, Rv0042c, Yz08 (15.6 kD)of Mycobacterium tuberculosis; Yz08 (15.6 kD) of Mycobacterium leprae;MTH313 of Methanobacterium thermoautotrophicum; Lrs14 of Sulfolobussolfataricus; CinR of Archaeoglobus fulgidus; PetP of Rhodobactercapsulatus; and SlyA (E293909) of Sinorhizobium meliloti.
 29. (canceled)